Toxins that modify targets within the cytosol of mammalian cells are crucial virulence factors in many bacterial diseases, such as anthrax. Such toxins must cross cellular membranes, and that process is not well understood for any bacterial toxin. Anthrax toxin consists of a pore-forming moiety, protective antigen (PA), and two enzymatic moieties, lethal factor (LF) and edema factor (EF). PA forms a heptameric pore in the endosomal membrane, which serves as a passageway for LF and EF to cross the endosomal membrane to the cytosol. A structure (the Phe-clamp), consisting of a heptad of solvent-exposed phenylalanine residues (F427) in the lumen of the PA pore, is essential for polypeptide translocation through the pore. The Phe-clamp interacts with translocating polypeptides and forms a seal against the passage of ions, which is believed to preserve the driving force for translocation, the transmembrane proton gradient. In addition, some F427 mutations are dominantly negative;that is, they co-oligomerize with wild-type (WT) PA to form nonfunctional pores. To study the dominant-negative mutations and to develop a platform for mapping translocating polypeptides within the PA pore, we have developed a protocol to purify a heteroheptamer consisting of one mutant and six WT subunits. As prototypes, we have constructed and partially characterized a heteroheptamer containing a single F427A or F427C subunit. We will use this technology to investigate the following specific aims: 1) Characterize variations in dominant-negativity of Phe-clamp mutations. The effects of various F427 mutations in a single subunit of the PA heptamer will be examined in both planar phospholipid bilayers and toxicity studies in cultured cells. 2) Map the interactions between a translocation substrate and the lumen of the PA pore. Using disulfide crosslinking in conjunction with electrophysiological measurements, we will determine the range of residues on LFN, the N-terminal domain of LF, that are accessible to F427C on a single subunit within the PA heteroheptamer under standard conditions. In a complementary approach, we will determine the range of residues in the pore that are accessible to Cys at the N terminus of LFN. PUBLIC HEALTH RELEVANCE Our research will advance understanding of a key step in the action of anthrax toxin and will contribute to the larger question of how proteins are translocated across membranes.